WO1999018981A1 - Implants comprenant des combinaisons de cellules allogeniques pouvant etre utilisees dans le traitement du cancer - Google Patents

Implants comprenant des combinaisons de cellules allogeniques pouvant etre utilisees dans le traitement du cancer Download PDF

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WO1999018981A1
WO1999018981A1 PCT/US1998/021413 US9821413W WO9918981A1 WO 1999018981 A1 WO1999018981 A1 WO 1999018981A1 US 9821413 W US9821413 W US 9821413W WO 9918981 A1 WO9918981 A1 WO 9918981A1
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cells
tumor
patient
human
lymphocytes
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PCT/US1998/021413
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English (en)
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James A. Thompson
Gale A. Granger
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The Regents Of The University Of California
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Priority to DE69837552T priority Critical patent/DE69837552D1/de
Priority to EP98952216A priority patent/EP1021197B1/fr
Priority to AU97969/98A priority patent/AU9796998A/en
Priority to CA002346769A priority patent/CA2346769C/fr
Publication of WO1999018981A1 publication Critical patent/WO1999018981A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/80—Neurotransmitters; Neurohormones
    • C12N2501/82—Histamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

  • astrocytomas glioblastoma multiforme
  • pancreatic cancer the fifth leading cause of cancer-related deaths in the United States The disease is associated with a high mortality rate, with a medium survival for untreated patients after diagnosis of about 4 months
  • chemotherapeutic agent Gemcitabine (GEM.ZARTM) was shown to improve overall median survival to 5 7 months compared to that of 5-fluorouracyl (4 2 months) and had a better clinical benefit index.
  • GEM.ZARTM the chemotherapeutic agent
  • An emerging area of cancer treatment is immunotherapy.
  • immunological strategies including: 1. Adoptive immunotherapy using stimulated autologous cells of various kinds; 2. Systemic transfer of allogeneic lymphocytes; 3.
  • Vaccination at a distant site to generate a systemic tumor-specific immune response 4. Implantation of immune cells directly into the tumor.
  • the first of these strategies is directed towards providing the patient with a level of enhanced immunity by stimulating cells ex vivo, and then readministering them to the patient.
  • the cells are histocompatible with the subject, and are generally obtained from a previous autologous donation.
  • One version is to stimulate autologous lymphocytes ex vivo with tumor-associated antigen to make them tumor-specific.
  • Zarling et al. (1978) Nature 274:269-71 generated cytotoxic lymphocytes in vitro against autologous human leukemia cells.
  • Osband suggests activating a tumor patient's mononuclear cells by culturing them ex vivo in the presence of tumor cell extract and a non-specific activator like phytohemagglutinin or IL-1, and then treating the culture to deplete suppresser ceil activity.
  • systemic administration of ex v/Vo-stimulated autologous tumor-specific lymphocytes has not become part of standard cancer therapy.
  • Autologous lymphocytes and killer cells may also be stimulated non-specifically.
  • Fc receptor expressing leukocytes that can mediate an antibody-dependent cell-mediated cytotoxicity reaction are generated by culturing with a combination of IL-2 and IFN- ⁇ (U.S. Patent No. 5,308,626).
  • peripheral blood-derived lymphocytes cultured in IL-2 form lymphokine-activated killer (LAK) cells, which are cytolytic towards a wide range of neoplastic cells, but not normal cells.
  • LAK cells have had some success in the treatment of metastatic human melanoma and renal cell carcinoma. Rosenberg (1987) New Engl. J. Med. 316:889-897.
  • TIL tumor-infiltrating lymphocytes
  • TILs can only be prepared in sufficient quantity to be clinically relevant in a limited number of tumor types, and remain experimental.
  • the second of the strategies for cancer immunotherapy listed earlier is adoptive transfer of allogeneic lymphocytes.
  • the rationale of this experimental strategy is to create a general level of immune stimulation, and thereby overcome the anergy that prevents the host's immune system from rejecting the tumor.
  • Strausser et al. (1981) J. Immunol. Vol. 127, No. 1 describe the lysis of human solid tumors by autologous cells sensitized in vitro to alloantigens.
  • Zarling et al. (1978) Nature 274:269-71 demonstrated human anti-lymphoma responses in vivo following sensitization with allogeneic leukocytes. Kondo et al.
  • tumor cells are genetically altered to produce a costimulatory molecule.
  • Tumor cells have been genetically altered to produce TNF- ⁇ , IL-1 , IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IFN- ⁇ , IFN- ⁇ and GM- CSF.
  • the vaccines comprise a source of tumor-associated antigen, particularly tumor cells from the patient to be treated, combined with an allogeneic cytokine-secreting cell line.
  • exemplary cytokines are IL-4, GM-CSF, IL-2, TNF- ⁇ , and M-CSF in the secreted or membrane-bound form.
  • the cytokine-producing cells provide immunostimulation in trans to generate a specific immune response against the tumor antigen.
  • Vaccines can be tailored for each type of cancer or for each subject by mixing tumor antigen with an appropriate number of cytokine-producing cells, or with a cocktail of such cells producing a plurality of cytokines at a favorable ratio.
  • the fourth of the immunotherapy strategies listed earlier is intra-tumor implantation, directed at delivering effector cells directly to the site of action. The proximity of the effector cells to the target is supposed to promote the ability of the transplanted cells to react with the tumor, generating a graft versus tumor response. Kruse et al (Proc.
  • the present invention provides compositions and methods for treating a tumor or eliciting an anti-tumor immunological response in a human patient.
  • the compositions contain a combination of cells that are allogeneic to the subject being treated, at least one of which has been alloactivated in culture.
  • the compositions are designed for implantation into the tumor bed of the patient, where they evoke a local reaction with a long-term beneficial effect on the tumor.
  • Certain embodiments of the invention relate to methods for preparing a pharmaceutical composition containing alloactivated human donor lymphocytes for treating a tumor in a human patient, comprising the steps of (a) coculturing lymphocytes from a first human donor allogeneic to the patient, and leukocytes from a second human donor allogeneic to both the first human donor and the patient, so as to alloactivate the lymphocytes; (b) harvesting the cells and preparing them for human administration at a time when they are effective in the treatment of the tumor.
  • the alloactivated cells are typically harvested from culture near the time of peak cytokine secretion, and are typically effective when given as a single dose
  • compositions prepared according to the aforementioned methods, in some forms containing approximately 2 x 10 9 to 2 x 10 10 alloactivated cells
  • the pharmaceutical compositions are suitable for human use after washing substantially free of substances like growth factors and serum inappropriate for administration, and in substantially sterile condition
  • the cell population contains leukocytes from at least three different humans
  • the cell populations can be used in a medicament for treatment of a tumor or raising an anti-tumor immune response in a human patient
  • the medicament is implanted at the site of a solid tumor, with or without prior resection or partial resection
  • Also embodied are methods for treating a tumor in a human patient or raising an anti-tumor immune response comprising implanting in or around the bed of a solid tumor in the patient a cell population comprising alloactivated human lymphocytes, the cell population having been produced by cocultunng lymphocytes from a first human donor ex vivo with leukocytes from a second human donor allogeneic to both the first human donor and the patient
  • the effect can optionally be boosted by implanting a second alloactivated cell population or administering a cellular vaccine
  • Potential benefits of administering the compositions of this invention include limiting the extent of tumor growth, improving quality of life, or extending the median life expectancy
  • Figure 1 is a bar graph showing the effect of different alloactivated lymphocyte preparations on providing resistance to a secondary challenge with J588L lymphoma cells in Balb/c mice Allogeneic cells stimulated either with syngeneic splenocytes or certain third-party splenocytes are both effective
  • Figure 2 is a bar graph showing the effect of different cell culture ratios on survival time in the mouse lymphoma model
  • Figure 3 is a bar graph showing the degree of functional activity in different human alloactivated cell preparations, as determined in four different assays
  • Figure 4 is a bar graph showing the level of secretion of the cytokines IL-2 and IFN- ⁇ by human alloactivated cell preparations
  • Figure 5 is a bar graph showing the enhancement of alloactivation of human lymphocytes by using a plurality of different stimulator cells
  • Figure 6 is a bar graph showing the degree of functional activity of different human alloactivated cell preparations, depending on the ratio of responder stimulator cells
  • Figure 7 is a bar graph showing the effect of including 20 ⁇ g/mL of histidine (dark shading) or cimetidine (light shading) into cultures of human cells, either the responder alone, the stimulator alone, or mixed cultures at a responder stimulator ratio of 10 1
  • This invention provides therapeutic compositions for use in cancer treatment
  • the compositions contain live cells, and confer a long-term benefit to human cancer patients when administered into a solid tumor mass
  • the results of an instructive experiment are shown in Figure 1
  • Balb/c mice were treated with a histocompatible lymphoma and an alloactivated cell population
  • host alloantigens C57 x Balb/c or Aj x Balb/c
  • a proportion of the mice clear the first dose of lymphoma cells
  • Some surviving mice are sufficiently well protected to survive a second challenge with the lymphoma ceils, consistent with ongoing specific immunity against tumor antigens
  • lymphocytes activated against alloantigens unrelated to those of the treated subject (C57 x Aj) are also effective in conferring survival from tumor challenge
  • cells from one donor can be alloactivated against alloantigens a second donor, and still be effective when administered into the tumor bed in a subject who is unrelated to either donor
  • the invention shares several features with the method for treating tumors described in
  • the live cells in the composition of the present invention include lymphocytes that are allogeneic to the subject being treated, and which have been alloactivated before use in treatment
  • the cells are implanted directly in or around a solid tumor mass in the patient with or without resection or partial resection of the tumor A key difference is the alloantigens that the lymphocytes in the composition have been activated against
  • the lymphocytes are activated using leukocytes of the patient to be treated, and are therefore primed specifically against the alloantigens of the patient
  • the lymphocytes are activated against alloantigens of a second unrelated donor
  • the donor is invariably allogeneic to the patient at a number of loci for both class I and class II histocompatibility antigens
  • the lymphocytes are typically not primed specifically against alloantigens of the intended recipient
  • Example 3 Preferred cell populations for use in this invention are those that show a high degree of alloactivation within the first three days, as measured by one or more of the screening assays This permits various donor donor cell populations to be screened in advance of use in therapy
  • a second strategy is to use a plurality of third party donors as a source of responder cells, stimulator cells, or both This is illustrated in Example 5 Using a plurality of donors helps ensure that at least some histoincompatibiiities will lead to sufficient alloactivation, as measured in the screening assays In addition, it has been found that cultures prepared with leukocytes from three or more donors can achieve higher overall levels of alloactivation
  • a third strategy is to include in the alloactivation culture an H2 receptor antagonist such as cimetidine This is also illustrated in Example 5
  • H2 receptor antagonists brings certain relatively inactive cell combinations over the threshold to measurable alloactivation, and increases the extent of alloactivation in others
  • the present invention confers a number of advantages in comparison with previously known technology For example, it is sometimes is difficult to get enough patient leukocytes to use as stimulators for preparing alloactivated cells
  • This invention provides that leukocytes from unrelated healthy donors can be used instead, thereby providing an almost limitless supply
  • particular donor combinations that generate high levels of alloactivation can be identified in advance, and used to provide a reliable source of effective material
  • Alloactivated cells may be stored or produced on an ongoing basis, eliminating the necessity of withholding treatment for the two to three days necessary to alloactivate lymphocytes using leukocytes of the patient.
  • MLC Multiple lymphocyte reaction
  • 'mixed lymphocyte culture', ' MLR", and “MLC” are used interchangeably to refer to a mixture comprising a minimum of two different cell populations that are allotypically different At least one of the aliotypically different cells is a lymphocyte
  • the cells are cultured together for a time and under suitable conditions to result in the stimulation of the lymphocytes
  • a frequent objective of an MLC is to provide allogeneic stimulation such as may initiate proliferation of the lymphocytes, but unless indicated, proliferation dunng the culture is not required
  • these terms may alternatively refer to a mixture of cells derived from such a culture
  • cytoimplant When cells from an MLC are administered as a bolus to a human, especially in a tumor bed, it is referred to as a "cytoimplant"
  • vacun immunological response
  • the immunological response may comprise antibodies, immunoreactive cells (such as helper/mducer or cytotoxic cells), or any combination thereof, and is preferably directed towards an antigen that is present on a tumor towards which the treatment is directed
  • a “cell line” or “cell culture” denotes higher eukaryotic cells grown or maintained in vitro It is understood that the descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell
  • Inactivation of a cell is used herein to indicate that the cell has been rendered incapable of cell division to form progeny
  • the cell may nonetheless be capable of response to stimulus, or biosynthesis and/or secretion of cell products such as cytokines
  • Methods of inactivation are known in the art Preferred methods of inactivation are treatment with toxins such as mitomycin C, or irradiation Cells that have been fixed or permeabilized and are incapable of division are also examples of inactivated cells
  • cancer cell refers to cells that have undergone a malignant transformation that makes them pathological to the host organism
  • P ⁇ mary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells
  • tumor-associated antigen refers to a molecule, complex, or epitope that is detected at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type
  • TM tumor-associated antigen
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of clinical pathology Desirable effects include preventing occurrence or recurrence of disease, alleviation of symptoms, dimimshment of any direct or indirect pathological consequences of the disease, preventing metastasis, lowering the rate of disease progression amelioration or palliation of the disease state, and remission or improved prognosis
  • the "pathology" associated with a disease condition is anything that compromises the well- being, normal physiology, or quality of life of the affected individual This may involve (but is not limited to) destructive invasion of affected tissues into previously unaffected areas, growth at the expense of normal tissue function, irregular or suppressed biological activity, aggravation or suppression of an inflammatory or immunological response, increased susceptibility to other pathogenic organisms or agents, and undesirable clinical symptoms such as pain, fever, nausea, fatigue, mood alterations, and such other features as may be determined by an attending physician
  • An "effective amount” is an amount sufficient to effect a beneficial or desired clinical result, particularly the generation of an immune response, or noticeable improvement in clinical condition
  • An immunogenic amount is an amount sufficient in the subject group being treated (either diseased or not) sufficient to elicit an immunological response, which may comprise either a humoral response, a cellular response, or both In terms of clinical response for subjects bearing a neoplastic disease, an effective amount is amount sufficient to palliate, ameliorate, stabilize, reverse or
  • An “individual” or “subject” is a vertebrate, preferably a mammal, more preferably a human.
  • Non-human mammals include, but are not limited to, farm animals, sport animals, and pets
  • the cellular compositions of this invention are prepared by ailoactivating one or more responder cell populations containing lymphocytes with one or more stimulator cell populations expressing alloantigens.
  • the source of the responder and stimulator cells are allogeneic both to . each other, and to the patient to be treated with the resultant composition.
  • Source of donor cells The cells that are used to prepare the composition are typically taken from healthy unrelated human donors allogeneic to the subject to be treated. Cells are generally described as allogeneic if they are from the same species but bear a phenotypic difference sufficient to stimulate an alloreaction. In the context of this disclosure, use of the term "allogeneic" is restricted to a difference in phenotype of major histocompatibility complex (MHC) antigens. Any qualitative difference in the identity of MHC allotypes between cells of the same species means they are allogeneic cells. In humans, differences at any of the HLA-A, B, C, D, DP, DQ, and DR loci constitute allotypic differences relevant for this invention. Identity of HLA-A, B, C, DP, DQ, and DR are typically determined using allotype-specific antibodies in a cytotoxicity or immunofluorescence technique.
  • MHC major histocompatibility complex
  • Preferred allotypic differences for the purposes of the present invention relate to HLA class II antigens. Comparing the class II antigens of the DP, DQ, and DR loci between the putative allogeneic cells and cells of the subject to be treated, preferably at least 1, and increasingly more preferably 2, 3, 4, 5, or even 6 loci are different between allogeneic cells. Class II antigens may also be determined at the D locus by mixed lymphocyte reaction using typed cells. Donors of allogeneic cells are generally unrelated to the subject being treated, to maximize the number of MHC mismatches. In a normal outbred population, unrelated individuals will almost invariably differ at a number of different loci.
  • Allogeneic cells are particularly suitable for use in the present invention if they demonstrate a strong proliferative response when tested in alloreactive cultures. Donors of cells previously known or empirically shown to produce a particularly strong response are especially suitable for use in therapy. As described elsewhere in this disclosure, a panel of different allogeneic cells can be tested in combinations to determine those that elicit the strongest degree of alloactivation.
  • the "responder" cells are capable of specifically reacting to an allogeneic stimulus.
  • the cell population generally contains lymphocyte cells or cells of the lymphocyte lineage, particularly T cells. Lymphocytes expressing CD4 antigen (CD4+ cells), and cells expressing CD8 antigen (CD8+ cells) are both included in the definition of T lymphocytes, and either or both may be included in the composition.
  • the responder cells are leukocytes obtained from peripheral blood, typically enriched for mononuclear cells (PBMC), and optionally further enriched for cells of the lymphocyte lineage
  • PBMC mononuclear cells
  • Particular enriched populations contain at least 10% CD4+ cells or 10% helper/inducer cells, more preferably they are at least about 20% of CD4+ or helper/inducer cells, even more preferably the portion is at least about 30% of CD4+ or helper/inducer cells
  • CD4+ cells may be conveniently quantified with commercially available specific antibody such as OKT4 in conjunction with fluorescence-activated counting
  • standard peripheral blood mononuclear cell preparations are suitably enriched for many applications of this invention Assays for determining the extent of alloactivation are described in the next section
  • the "stimulator" cells are allogeneic to the responder cells and capable of eliciting an alloreaction in the responders
  • Suitable cell types for use as stimulator cells are those that bear a high density of allogeneic histocompatibility antigens, particularly class II antigens Any type of cell (not limited to blood cells) bearing sufficient alloantigens can be used
  • a particularly suitable source is peripheral blood leukocytes or white cells It is desirable to enrich for or at least not to deplete cells expressing class II histocompatibility antigens from the population such as B cells and monocytes Extensive subfractionation of the cells is not usually required and a simple peripheral blood mononuclear cell population (PBMC) is adequate for most purposes
  • PBMC peripheral blood mononuclear cell population
  • the combined cell population is not necessarily restricted to one source for the responder cells and one source for the stimulator cells
  • Two, three, for, or a higher plurality donors may optionally be used to facilitate collection of the allogeneic cells, to increase stimulation of the allogeneic ceils, to minimize the ehcitation of an anti-allotype response, or to otherwise enhance the therapeutic efficacy
  • Donors are typically prescreened to identify those with sufficient leukocyte count, and exclude those with neoplastic conditions or transmissible infections Collection may be performed by whole blood donation followed by separation of blood cell populations, or by leukapheresis Leukapheresis is especially appropriate for collecting the responder cell population, because the number of cells required is substantial Sufficient blood is processed to obtain about 100-500 mL leukapheresis suspension, preferably at least about 200 mL
  • leukapheresis may be performed using a Cobe 2997 (COBE SPECTRA®, Lakewood CO), Fenwall CS 300 (Fenwall, Deerfield IL), or Haemonet ⁇ cs (Braintree MA) blood cell separator Flow rates of ⁇ 40-50 mUmin for 2-4 h yield ⁇ 200-250 mL leukapheresis suspension having ⁇ 1 mL red cells, with variations between individual donors and the equipment used
  • the collected leukocytes are generally washed to remove platelets, and resuspended in a suitable medium, such as AIM V supplemented with 2% inactivated fetal calf serum Separation of PBMC and other enrichment procedures include cent ⁇ fugation over a suitable medium such as FICOLLTM or HISTOPAQUE®, passage over a nylon-wool column, affinity separation methods such as panning, or sorting in a fluorescent cell sorter using an antibody against a relevant cell-surface marker Where possible, it is generally preferable to decrease the number of manipulation steps For example, better leukapheresis separation may obviate the need for subsequent separation on FICOLLTM
  • the ratio of responder stimulator cells is preferably between about 100 1 to 1 10, more preferably about 50 1 to 1 1 , still more preferably about 20 1 to 5 1, and even more preferably about 10 1
  • the ratio may be approximately 9 (1 1)
  • the ratio may be approximately 8 (1 1 1)
  • the ratio may be (5 5) 1 or (3 3 3) 1 If cultured together the multiple responder composition becomes a multi-way MLC
  • One-way activation of multiple responders can be achieved by conducting a separate culture for each responder population at a 10 1 ratio and then combining the alloactivated cells just before use
  • This invention encompasses the use of two-way or multi-way mixed lymphocyte cultures, wherein a plurality of cell populations act as both responders and stimulators
  • one-way MLCs are performed by inactivating the stimulator cells, for example, by treating ⁇ 10 7 cells/mL with 50 ⁇ g/mL mitomycin C or sublethal irradiation, followed by washing
  • the cells cultured at an appropriate density in a suitable atmosphere (such as 95% 0 2 , 5% C0 2 at about 37°C)
  • the culture period is preferably at least about 12 h more preferably between about 24 h and 72 h Additional stimulation may be obtained by culturing for 3-5 days although this is generally not preferred since cytokme levels are normally higher during the first 48 to 72 h of culture
  • cytokines can be tested in a standard immunoassay
  • Particular cytokines of interest are IL-2, IL-4, IL-6, TNF- ⁇ , LT, IFN- ⁇ , G-CSF, M-CSF (both membrane and secreted form), and GM- CSF
  • particular degrees of stimulation is indicated by levels of biological activity of TNF- ⁇ or LT at 50-150 U/mL, or 500-3500 pg/mL
  • Proxies for functional activity of the alloactivated ceils include I MTT Formazan Reduction Assay, II XTT Formazan Reduction Assay, III Flow Cytometry for CD3/CD69 or CD3/FDA, IV FDA Plate Assay, V Acid Production Assay, VI Acndine Orange Assay These assays are detailed in Example 3 More traditionally, alloactivation can be determined by cell proliferation, measured by culturing a test sample for 5 days and conducting a standard [ 3 H]-thym ⁇ d ⁇ ne uptake assay, or by counting blast cells The predictive value of functional assays can be determined by compa ⁇ ng results of the assays on cultured cells with the effect of the cells in a suitable animal model See Example 4
  • Preferred cultures are those that show a level of activation > 10% above unstimulated donor control value within one of the first 3 days of culture, as measured by the Tetrazolium Reduction Assay (XTT), the Acndine Orange Assay (AO) or by Flow Cytometry (CD69), more preferably attaining the threshold in several of these assays in combination
  • donors can be chosen according to experience, both in terms of the degree of alloactivation observed in culture, and the clinical result Functional criteria indicating a particular level of activation, such as the Tetrazolium Reduction Assay (XTT), Flow Cytometry Assay, or the level of secretion of certain lymphokmes determined by ELISA, may be sufficiently predictive of outcome, depending on clinical experience
  • XTT Tetrazolium Reduction Assay
  • Flow Cytometry Assay or the level of secretion of certain lymphokmes determined by ELISA
  • donors can be selected, if desired, on the basis of tissue match
  • Donors of particular human histocompatibility types can be tested for efficacy with particular tumors, if desired, using one of the chime ⁇ c animal models listed earlier
  • a more immediate donor identification test can be conducted using PBLs from the patient and PBL from a selection of potential donors in an in vitro assay
  • One such assay is a reverse functional test
  • patients cells are set up in a mixed lymphocyte culture as the responder, using the potential donor of the alloactivated cells as the inactivated stimulator Since the response is thought to involve cytokme secretion by the alloactivated cells, an alternative predictor may be a two-stage culture
  • a responder stimulator culture is set up using the same responder and stimulator cells being tested for use in the preparative culture At 3 days, the culture is inactivated with mitomycin or sub-lethal irradiation, so that cells can still produce cytokines but not replicate Leukocytes from the patient are then added, and their response is followed by a functional assay, cytokme secretion, or T cell proliferation
  • inactivated tumor cells are also provided in the second stage of the culture, and read-out is determined at
  • the degree of alloactivation or the potential therapeutic outcome can be enhanced by employing either of the following strategies a) using a plurality of donor cells as the responder or stimulator in the MLC and/or b) adding an H2 receptor antagonist to the culture medium of the MLC
  • H2 receptor antagonist inhibits the activity of suppressor T cells in the culture
  • a preferred H2 receptor antagonist is cimetidme, added to the culture medium at between 5 ⁇ g/mL and 100 ⁇ g/mL, typically 20 ⁇ g/mL
  • compositions of this invention can be administered to subjects, especially human subjects They are particularly useful for eliciting an immune response against a tumor-associated antigen, or for treating cancer
  • One purpose of implanting the cellular compositions of this invention is to elicit an immune response
  • the immune response may include either humoral or cellular components, or both Humoral immunity can be determined by a standard immunoassay for antibody levels in a serum sample from the treated individual
  • a cellular immune response is a response that involves T cells, and can be observed in vitro or in vivo
  • a general cellular immune response can be measured as the T cell pro ferative activity in ceils (particularly PBL) sampled from the subject after administration
  • Inactivated tumor cells preferably derived from the subject, are used as stimulators
  • a non-specific mitogen such as PHA serves as a positive control
  • incubation with an unrelated stimulator cell serves as a negative control
  • [ 3 H]thym ⁇ d ⁇ ne incorporation is measured
  • determination of which subset of T cells is proliferating can be performed using flow cytometry T cell cytotoxicity (CTL) can also be measured
  • CTL flow cytometry T cell cytotoxicity
  • an enriched T cell population from the subject are used as effectors in a standard 51 Cr release assay
  • Tumor cells are radiolabeled as targets with about 200 ⁇ Ci of Na 2 51 Cr0 4 for 60 minutes at 37° C, followed by washing T cells and target cells ( ⁇ 1 * 10 4 /well) are then combined at various effector-to-target ratios in
  • Another purpose of implanting the cellular compositions of this invention is for treatment of a neoplastic disease, particularly cancer Beneficial effects are typically immunologically mediated or the result of an inflammatory infiltrate into the injection site and collateral tumors
  • Evidence of a host response can be shown inter alia by infiltration of host leukocytes (such as lymphocytes, histiocytes, and other leukocytes) into the tumor site by standard histomorphology analysis
  • the response is preferably an immunological response, which may have humoral or cellular components, and preferably includes cytotoxic T cell activity Immunological activity can be measured systemically in standard antibody binding immunoassays or cytotoxicity assays on peripheral blood components taken from the treated subject, using tumor cells as targets Monitoring the effect according to these methods is optional, and the recited features need not be positively demonstrated in order for the compositions and treatment methods to fall within the scope of this invention, except where required
  • compositions of this invention may be used for administration to both human and non-human vertebrates Typically, the subject will either have cancer, or be at substantial risk of developing cancer
  • Typical human subjects for therapy comprise two groups, which may be distinguished by clinical cntena Patients with "advanced disease” or “high tumor burden” are those who bear a clinically measurable tumor A clinically measurable tumor is one that can be detected on the basis of tumor mass (e.g., by palpation, MRI, CAT scan, X-ray, or radioscintigraphy; positive biochemical or histopathological markers on their own are insufficient to identify this population).
  • tumor mass e.g., by palpation, MRI, CAT scan, X-ray, or radioscintigraphy; positive biochemical or histopathological markers on their own are insufficient to identify this population.
  • a cellular composition for use in this invention is administered to patients with advanced disease with the objective of palliating their condition. Ideally, reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit. Clinical improvement includes decreased risk or rate of progression or reduction in pathological consequences of the tumor.
  • a second group of suitable subjects is known in the art as the "adjuvant group". These are individuals who have had a history of cancer, but have been responsive to another mode of therapy.
  • the prior therapy can have included (but is not restricted to) surgical resection, radiotherapy, traditional chemotherapy, and other modes of immunotherapy.
  • these individuals have no clinically measurable tumor by the definition given above.
  • they are suspected of being at risk for recurrence or progression of the disease, either near the original tumor site, or by metastases.
  • the adjuvant group may be further subdivided into high-risk and low-risk individuals.
  • the subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts, and are suitably defined for each different cancer.
  • a cellular composition for use in this invention is administered to patients in the adjuvant group in order to elicit an anti-cancer response primarily as a prophylactic measure against recurrence.
  • the composition delays recurrence of the cancer, or more preferably, reduces the risk of recurrence (i.e., improves the cure rate).
  • Such parameters may be determined in comparison with other patient populations and other modes of therapy.
  • the cellular compositions can be administered at any time that is appropriate.
  • therapy can be conducted before or during traditional therapy of a patient with high tumor burden, and continued after the tumor becomes clinically undetectable. Therapy may be continued in a patient who initially fell in the adjuvant group, but is showing signs of recurrence.
  • the list includes sites that are thought to be immune privileged, such as the brain, and sites that are not immune privileged, such as the pancreas, colon, breast, and prostate.
  • Brain tumors such as astrocytoma, oligodendroglioma, ependymoma, medulloblastomas, and PNET (Primitive Neural Ectodermal Tumor);
  • Pancreatic tumors such as pancreatic ductal adenocarcinomas.
  • Lung tumors such as small and large cell adenocarcinomas, squamous cell carcinoma, and bronchoalveolarcarcinoma;
  • Colon tumors such as epithelial adenocarcinoma, and liver metastases of these tumors;
  • Liver tumors such as hepatoma, and cholangiocarcinoma
  • Breast tumors such as ductal and lobular adenocarcinoma
  • Gynecologic tumors such as squamous and adenocarcinoma of the uterine cervix, and uterine and ovarian epithelial adenocarcinoma,
  • Prostate tumors such as prostatic adenocarcinoma
  • Bladder tumors such as transitional, squamous ceil carcinoma
  • Tumors of the RES System such as B and T cell lymphoma (nodular and diffuse), plasmacytoma and acute and chronic leukemia,
  • Skin tumors such as malignant melanoma
  • Soft tissue tumors such as soft tissue sarcoma and ieiomyosarcoma
  • the immune status of the individual may be any of the following
  • the individual may be immunologically naive with respect to certain tumor-associated antigens present in the composition, in which case the compositions may be given to initiate or promote the maturation of an anti-tumor response
  • the individual may not currently be expressing anti-tumor immunity, but may have immunological memory, particularly T cell memory relating to a tumor-associated antigen, in which case the compositions may be given to stimulate a memory response
  • the individual may also have active immunity (either humoral or cellular immunity, or both) to a tumor-associated antigen, in which case the compositions may be given to maintain, boost, or maturate the response, or recruit other arms of the immune system
  • the subject should be at least partly immunocompetent, so as to minimize a graft versus host reaction of pathological scope
  • cancer patients often show a degree of immunosuppression, and this does not
  • compositions of this invention can be administered to the subject at the site of any solid tumor Circulating cancers are treatable so long as there is at least one solid tumor mass Metastatic sites, affected nodes, and other sites away from the primary neoplasm are suitable, so long as they are accessible and contain sufficient tumor antigen
  • the composition can be administered at or near the site or in a cavity created by the resection If the tumor is completely removed, however then it may be preferable to administer the alloactivated cells to a metastatic site to increase the local amount of bystander tumor antigen
  • the most convenient time to administer the alloactivated cells to a resectable site is during the time of surgery To keep the cells at the site until completion of the surgical procedure, it is convenient to administer the cells in a pharmaceutically compatible artificial gel, or in clotted plasma
  • the composition can be injected at or near the tumor site through a needle
  • the needle can be positioned using ultrasound, radioscintigraphy, or some other imaging technique, alone or in combination with the use of an appropriate scope or cannula
  • Pancreatic tumors are preferably implanted using an injection needle positioned by an endoscopic ultrasound guided technique, as described by Chang et
  • the cell population is conveniently administered when suspended in isotonic saline or a neutral buffer to a volume of about 10 mL.
  • the dose given is an amount "effective" in bringing about a desired therapeutic response, be it the stimulation of an immune response, or the treatment of cancer as defined elsewhere in this disclosure.
  • effective doses typically fall within the range of about 10 8 to 10 11 cells, including allogeneic stimulators and responders.
  • Multiple doses when used in combination to achieve a desired effect each fall within the definition of an effective amount.
  • the various components of the implant composition are present in an "effective combination", which means that there are sufficient amounts of each of the components for the composition to be effective.
  • at least about 10 8 more preferably between about 1 x 10 9 to 5 x 10 10 and; more preferably between about 2 10 9 to 2 10 10 responder cells are present.
  • at least about 10 7 more preferably between about 5 ⁇ 10 7 to 5 ⁇ 10 9 and; more preferably between about 1 10 8 to 2 » 10 9 stimulator cells are present.
  • Ratios of allogeneic lymphocytes to stimulator leukocytes is generally between 1 :1 and 100:1 , usually between about 5:1 and about 25:1 , and typically about 10:1.
  • any number of component cells or other constituents may be used, as long as the composition is effective as a whole. This will also depend culture conditions and other factors during preparation.
  • compositions of this invention may be given following, preceding, in lieu of, or in combination with, other therapies relating to generating an immune response or treating cancer in the subject.
  • the subject may previously or concurrently be treated by chemotherapy, radiation therapy, and other forms of immunotherapy and adoptive transfer. Where such modalities are used, they are preferably employed in a way or at a time that does not interfere with the immunogenicity of the compositions of this invention.
  • the subject may also have been administered another vaccine or other composition in order to stimulate an immune response.
  • Such alternative compositions may include tumor antigen vaccines, nucleic acid vaccines encoding tumor antigens, anti-idiotype vaccines, and other types of cellular vaccines, including cytokine-expressing tumor cell lines.
  • Certain embodiments of this invention relate to combination therapies.
  • the subject is given an intra-tumor implant of stimulated allogeneic lymphocytes, either before, during, or after treatment at a site distant from the tumor with a composition comprising stimulated allogeneic lymphocytes and autologous tumor cells.
  • a composition comprising stimulated allogeneic lymphocytes and autologous tumor cells.
  • certain embodiments of this invention relate to administering a cytoimplant, and subsequently boosting the therapeutic effect or immunological response by administering to the patient a composition comprising alloactivated human lymphocytes allogeneic to the patient and an inactivated cell population consisting of tumor cells from the patient or progeny thereof
  • compositions of this invention are generally effective when given at a single dose, it may be desirable to readminister the composition at intervals of 3-6 months, especially for fast-growing tumors that can be injected through a positioned needle
  • certain embodiments of this invention relate to administering a cytoimplant, and subsequently boosting the therapeutic effect or immunological response by implanting in or around the bed of a solid tumor in the patient a second cell population comprising alloactivated human lymphocytes allogeneic to the patient
  • Timing of administration of compositions of this invention is within the judgment of the managing physician, and depends on the clinical condition of the patient, the objectives of treatment, and concurrent therapies also being administered
  • Suitable means of immunological monitoring include a one-way MLR using patient's PBL as responders and primary tumor cells as stimulators
  • An immunological reaction may also be manifest by a delayed inflammatory response at the injection site
  • Suitable means of monitoring of the tumor are selected depending on the tumor type and characteristics, and may include CT scan, magnetic resonance imaging (MRI), radioscinti
  • EXAMPLE 1 MIXED LYMPHOCYTE CULTURE PROCEDURE.
  • PBMCs Peripheral blood mononuclear cells
  • CBC complete blood count
  • leukapheresis suspension containing PBMC was collected from each donor, using standard blood donation procedures for supportive apheresis according to the manufacturers' instructions.
  • the leukapheresis was performed using a Fenwall CS 3000 (Deerfield, EL) blood cell separator.
  • a flow rate of 40 to 50 ml/min for 2 to 4 hours with lymphocyte yield of 2-4 x 10 9 processed a total donor blood volume of 7,000 to 12,000 ml to yield 200 to 250 ml of leukapheresis suspension having less than 1 mi of red cells.
  • the centrifuge rate was 5 x g
  • the flow rate was up to 45 ml/min
  • the collection rate was no more than or equal to 2.5 ml/min.
  • Cells were drained from the leukapheresis pack into two or three 250 ml centrifuge tubes, removing and setting aside 3 ml for sterility tests to be done during centrifugation. Cell concentrate was diluted with phosphate buffered saline (PBS) and centrifuged for 7 minutes at 2,000 rpm.
  • PBS phosphate buffered saline
  • Centrifugation was repeated twice for a total of three times to wash the cells free of the clotting factor in the donor's serum.
  • the mononuclear cells at the interface between the Histopaque® cell separation medium and the plasma layer were carefully collected with a 25 sterile pipet into 2 sterile 250 ml centrifuge tubes and diluted with 2% AIM V to a final volume of 250 ml
  • the diluted mononuclear cells were centnfuged at 550g for 7 to 10 minutes
  • the supernatant was discarded, then the cell pellet was re-suspended with 2% AIM V and centnfuged at 550g for 5 minutes
  • the washing step was repeated for a total of three times
  • cells were re-suspended in 50 ml of 2% AIM V 1 ml of the cell suspension was diluted 1 10 in 2% AIM V per tube, and the number of viable cells was determined by enumeration in a 1 1 in Trypan Blue as described above
  • Alloactivation The isolated patient PBMCs were re-suspended at 10 7 cells/ml in AIM V, 50 ⁇ g Mitomy ⁇ n C (Bristol-Mayer Squibb, Princeton, NJ) were added per ml of patient cell suspension, and the suspension of PBMCs was incubated at 37°C for one hour to block response of the stimulator cells to the responder cells After one hour of incubation, the excess mitomycm C was washed from the cells by alternate centrifugation (250g for 5 mm), and the cells were resuspended in AIM-V After mitomycm treatment of the patient's PBMCs the cells were added at a 20 1 to 10 1 donor cell patient cell ratio to the donor culture)
  • the donor and mitomycm C-treated patient PBMC suspension was placed in a sealed sterile Fenwal tissue culture system especially designed for culture of PBMC for reimplantation into patients Cells were passed in sealed systems via Fenwal cell transfer units and pumps according to the manufacturers instructions, and cultured in a 37°C incubator for 48 hours
  • Sterility testing of alloactivated cells Two days prior to implantation of the ceil suspension, the following three sterility tests were performed 10 mi sterile aliquots were removed from each tissue culture bag, placed into sterile capped 15 ml centrifuge tubes, and centnfuged for 10 minutes at 450g In each tube, the pellet was resuspended in 3 0 ml of PBS A 1 ml aliquot of the cell suspension was added to each of three sterile capped tubes containing 2 ml of thioglycollate broth, tryptic soy broth, or RPMI-10% and incubated for 48 hours Each cell suspension was examined microscopically prior to implant to detect signs of microbial growth
  • the cells were centnfuged out of their medium, washed two times with saline and re-suspended in platelet free, decalcified plasma obtained from the patient the previous day The cells were transported to the operating room in plasma, then the plasma was re-calcified by the addition of calcium giuconate so that it clots just before implantation into the tumor bed
  • cytoimplant cells The procedure for preparing the cytoimplant cells was generally in accordance with the main features of Example 1. Typically, a volunteer third-party donor for responder cells is screened by normal blood bank criteria for suitability. No special matching or identification of HLA type is performed. Whole blood or leukapheresis is collected from the patient to be treated; and leukapheresis is collected from the donor on the same day. Mononuclear cells are prepared from both patient and donor by centrifugation on FicollTM, and counted to ensure that enough cells are present to prepare the intended dose. Patient cells are inactivated by treating for 1 hour with mitomycin C, and then washed.
  • the cells are combined at a dono patient ratio of 10:1 to 20:1 , depending on the number of patient cells available.
  • the cells are suspended at 3 x 10 6 per mL in AIM 5 medium containing 2% fetal calf serum and antibiotics in a gas-permeable plastic bag, and incubated at 37°C in an atmosphere of 5% C0 2 /95% 0 2 . No cytokines or other growth factors are added. After three days, the cells are collected by centrifugation and washed. The cells are then transferred to the clinic in a medium suitable for administration. For the treatment of pancreatic cancer, the cells were suspended in a volume of about 10 mL isotonic saline.
  • the treatment was conducted as follows. A sufficient amount of whole blood or leukapheresis was collected from each patient to prepare the cultured cells used in treatment. The sample was forwarded to the Immunotherapy Lab, and used to prepare stimulator cells for allogeneic stimulation of third-party lymphocytes
  • the cytoimplant cells were administered to the subject on an out-patient basis. Under light anesthesia, an injection needle was positioned into the tumor using an endoscopic ultrasound guided technique The implant cells were rescued from culture, washed, suspended in about 10 mL of injectable isotonic saline, and delivered to the diagnostic service center. The cells were injected into the tumor mass, the device was removed, and the patient was allowed to recover Three patients were administered with a single dose of 3 x 10 9 implant cells. Four patients were administered with a single dose of 6 x 10 9 implant cells. Three patients were administered with a single dose of 9 x 10 9 implant cells.
  • Patient 001 was a 78 year old male with an unresectable clinical Stage IV tumor. The patient received treatment on a compassionate basis, and survived 6.5 months.
  • Patient 002 was a 53 year old female with an unresectable Stage III tumor. The patient later presented with elevated total bilirubin and died at 4 2 months after developing liver metastasis
  • Patient 003 was a 60 year old male with unresectable Stage III tumor There was a hospital admission for synocopal episode The patient survived 20 8 months
  • Patient 004 was a 52 year old male with an unresectable Stage II tumor The patient was later admitted to hospital with biliary obstruction, cholangitis, and dehydration This patient died 20 7 months after treatment
  • Patient 005 was an 89 year old female with a Stage I tumor, who was not a candidate for resection due to her age She received treatment on a compassionate basis, and died 11 3 months later due to myocardiai infarction
  • Patient 006 is a 54 year old female with an unresectable Stage III tumor She was later admitted to hospital for intractable nausea, vomiting and dehydration, and subsequently for gastrointestinal hemorrhage There was increased tumor size and liver metastasis The patient died 4 3 months after treatment
  • Patient 007 is a 61 year old female with an unresectable Stage II tumor On follow-up, there was elevated total bilirubin, and the patient was admitted with intractable nausea and vomiting, diarrhea, and dehydration possibly related to colitis flare-up The patient is still alive >
  • Patient 008 is a 55 year old female with an unresectable Stage IV tumor No serious adverse events were observed, and the patient is still alive >13 months after treatment
  • Patient 009 is a 54 year old male with an unresectable Stage II tumor The patient was later admitted for two days for pain and nausea and vomiting The patient died 11 7 months after treatment
  • Patient 010 is a 68 year old male with an unresectable Stage II tumor No serious adverse events were observed, and the patient died 8 5 months after treatment
  • One photomicrograph showed fibrovascular tissue with scattered individualized tumor cells. There is a dense iymphocytic and plasma cell infiltrate. Another field showed lymphocytes rosetting the separated tumor cells. The tumor cells were dark and shrunken, which is evidence of apoptosis. Another field showed scattered islands of necrotic tumor cells. There was a very dense infiltrate of lymphocytes, and lymphocytes appear to be trafficking into the site from adjacent venules. In a high magnification view there was clear evidence of direct contact between lymphocytes and necrotic tumor cells.
  • the histomorphology analysis provide clear evidence of a local response by cells of the patient after implantation of the alloactivated cells.
  • the data are consistent with the cell response in the patient having a direct role in the beneficial effects of the treatment, as shown by direct contact between lymphocytes and necrotic tumor cells. To the limited extent that infiltrating cells are present in untreated pancreatic cancer, this type of direct contact is not observed.
  • a method of measuring the potency of the alloactivated cells can be employed. Only cell cultures with activity over and above unstimulated control cells should be used clinically. It is beneficial to compare the activity to the unstimulated control, since baseline activity of mononuclear cells from different individuals varies widely.
  • lymphocyte activation Compared with unstimulated mononuclear cells, alloactivated cells reduce more Formazan dye and have more esterase activity. Turnover of XTT (a Formazan dye) can be easily demonstrated in a 96-well plate by colorimetric spectrophotometry at 470 nm (reference 650 nm). Activated cells typically show higher absorbance than controls. Lymphocyte activation can also be demonstrated by flow cytometric determination of esterase activity using the esterase substrate, fiuorescein diacetate (FDA). T cells with high esterase are not determined using FDA and a Phycoerythrin-labeled CD3 antibody.
  • FDA fiuorescein diacetate
  • Esterase activity can be accurately measured in a plate assay by using higher concentrations of FDA and determination of esterase activity by spectrophotometry at 494 nm (reference 650 nm) in a 96-well plate format. Background esterase activity inherent to serum- containing media is inhibited by addition of a competitive esterase inhibitor ( ⁇ 10 mM), arginine methyl ester. For the most part, these measures show good correlation with each other and with blastogenesis.
  • a competitive esterase inhibitor ⁇ 10 mM
  • This assay is used to enumerate live cells by ability for culture sample to reduce MTT to blue-green Formazan dye, and is also helpful for the distinguishing activated from inactive cells. It can be used for practically any cell in practically any media
  • the useful cell range is between 10 5 and ⁇ x 10 6 per mL
  • This assay is used to enumerate live cells by ability for culture to sample to reduce XTT to red-orange Formazan dye, and is also helpful for the distinguishing activated from inactive cells It can be used for practically any cell in practically any media
  • the useful cell range is between 10 5 and 5 x 10 6 per mL
  • Test samples of donor and patient cells are mixed in small cultures at 0 5 x 10 6 cells/mL in 2% FCS-RPMI These cultures are maintained at 37°C in 5% C0 2 incubator until testing
  • CD3-PE Coulter
  • CD69-FITC Becton-Dickmson
  • Mononuclear cells from a normal donors can be used to produce activated control specimens. These cells are placed in 2% FCS-RPMI at 0.5 x 10 6 cells/mL up to 100 mL. Cells are cultured for 2 days at 37°C in the presence or absence of 2 ⁇ g/mL PHA lectm, or admixed at a ratio of 10:1 with a second donor population The cells are collected by centrifugation at 350 X g for 5 minutes. The media is removed and replaced by 1/10th the volume of DMSO Freezing media, and frozen When needed, control unstimulated and stimulated cells can be thawed quickly and resuspended at the original volume by adding 9 volumes of PBS. Control cells are analyzed according to the protocol below along with samples from the test culture The duplicate use of control specimens is an addition quality assurance measure The percentage of CD69 or esterase positive lymphocytes should be within a 5% variance
  • Coulter Flow Cytometer Histogram 1 (forward scatter vs CD3) of either protocol should have a generous gate around the CD3+ mononuclear cells Region A should approximate % T- Lymphocytes and should be passed to Histogram 2
  • Histogram 2 the use of side scatter versus CD3 permits discrimination of lymphocytes (low side scatter level) from unlysed RBSs, RBC ghosts, platelet aggregates, residual granulocytes and/or other debris
  • a gate is drawn around the lymphocytes (see Histogram 2 for example)
  • This second gate is passed to Histogram 3, where the CD3+ CD69+ cells or CD3+ FDA+ cells are displayed Run the control values first to set gates (unstimulated controls) Place the quad stat cursor of Histogram 3 so that the CD69 or FDA high values (Quad 2) are 2% Leave this gate set when analyzing stimulated samples Count at least 5,000 gated cells for each sample to obtain a 97% confidence interval
  • This assay is used to enumerate live cells by ability for culture sample to turnover the esterase substrate, fluorescein diacetate, and is also helpful for the distinguishing activated from inactivated cells
  • This assay can be used for practically any media
  • the useful cell range is between 10 5 and 5 x 10 6 per mL
  • This assay is used to quantitative relative organic acid production in cultures. This correlates with the state of activation of cells. This assay requires the use of medium containing no more than 2% serum. Practical cell range is 1-5 x 10 6 cells/mL incubated from 24-48 hours.
  • Acid Analysis Reagent This is made in bulk and stored at 4°C. Add 0.1 mg/mL Bromophenol Blue in distilled water. Add sufficient concentrated HCI until the appropriate titration point is met. Titration is performed until yellow-green color is obtained after adding 75 ⁇ L of reagent to 100 ⁇ L RPMI 2% FCS in a well of a 96 well plate.
  • This assay is used to quantitate the absolute number of lymphoblasts produced in cultures after 7 days.
  • the useful cell range is between 1 x 10 5 and 5 x 10 6 per mL.
  • spleen cells are cultured in 5 x 75 mm polypropylene tubes identical to the AO test. After 7 days at 37 C, the cells are mixed by vortexing and a cytospin preparation is made (Shandon cytocentrifuge, ). The slides are stained with Wright/Giemsa stain using an automated slide stainer and the blasts enumerated manually by counting at least 300 cells/slide. The percent blasts is calculated by dividing the number of blasts by the total number of nucleated ceils.
  • Stimulator spleen cells are then prepared identical to responder spleen cells but are irradiated with 3000 R (Cs 137 source) prior to use
  • 3000 R Cs 137 source
  • One hundred ul of the stimulator cells are added and the mixed lymphocyte culture is incubated at 37 C for 7 days in a 95% a ⁇ r/5% C0 2 atmosphere
  • 10 ul of H hymidine 5 mCi/ml, ICN Pharmaceuticals, Costa Mesa, CA
  • the microtiter plate is then harvested used a MASH harvestor and the amount of incorporated thymidine determined by counting the harvested wells in a liquid scintillation counter
  • the stimulation index (SI) is then determined by calculating the ratio of the CPM of H 3 -Thym ⁇ d ⁇ ne incorporated into the MLC culture divided by the CPM of H 3 -thym ⁇ d ⁇ ne incorporated into the control (unstimulated) culture
  • Splenocytes form the mice were used to produce the alloactivated cells by culturing at a ratio of 10 1 responder stimulator cells Splenocyte combinations were cultured in RPMI plus 10% fetal calf serum (FCS) supplemented with penicillin-streptomycin at 3 x 10 6 /mL at 37°C for 3 days 1 x 10 6 live J588L lymphoma cells were admixed with 10 x 10 6 cultured mouse splenocytes, and then injected into the subcutaneous tissue over the right flank of Balb/c mice Treated mice were watched for tumor growth for 3 weeks
  • Figure 2 shows the percentage of mice without tumors after primary tumor challenge (6 mice per group) A lower cell ratio may on some occasions be better at inducing an antitumor response in mice
  • Splenocytes were taken from naive C57 or Balb/c mice or from a mouse bearing a 1 cm lymphoma in the right flank The cells were cultured for 3 days either alone or after admixture with
  • Balb/c cells at a 10 1 ratio at a concentration of 0 5 x 10 s cells/mL in RPMI-10% FCS Lymphocyte activation was judged by analyzing the percentage of CD3+/Esterase high population by Flow
  • mice were admixed with live lymphoma cells (J588L cells) and injected into the flanks of naive Balb/c mice The mice were monitored for tumor growth for 3 weeks Mice without tumors were next rechallenged with 1 x 10 6 live lymphoma cells alone in the left flank, and watched for tumor growth Percent mice without tumors after secondary tumor challenge was between 30 and 40% in both groups being slightly higher in the group treated with cells stimulated using naive Balb/c donors
  • C57/BL6 mice (3 per group) were injected subcutaneously with 10 6 irradiated B16 melanoma cells alone, mixed with 10 7 Balb/c x C57 alloactivated lymphocytes, or mixed with 10 6 IL-4 secreting J588L lymphoma cells (allogeneic to C57)
  • the alloactivated cells were prepared by culturing Balb/c splenocytes with C57 splenocytes at a ration of 10 1 at 3 x 10 6 /mL in RPMI 10%
  • mice were washed in PBS, and injected subcutaneously in the flanks of naive C57 mice After 3 weeks, the mice were rechallenged with 5 x 10 5 B16 live melanoma cells subcutaneously in the opposite flank Mice were observed for tumor formation and sacrificed after tumors reached 1 cm in diameter The mice treated with the alloactivated cells survived significantly longer than the other groups The two longest surviving mice finally developed cone-shaped tumors, both of which ulcerated No other mice developed ulcers Two days after the ulcers appeared, both mice expired Necropsy of these mice revealed the presence of extremely necrotic tumor cells, with evidence of recent tumor cell lysis in the form of massive DNA deposition This necrosis was accompanied by an inflammatory infiltrate, consisting mostly of lymphocytes No other form of infection was observed anywhere in the body No lung metastases were seen This is in contrast to the large number of lung metastases in control mice harboring B16 melanoma tumors in the flank Bilateral kidneys in both mice showed extensive glomerulonep
  • C57/BL6 mice (3 per group) were injected subcutaneously with 10 6 Lewis Lung carcinoma cells alone, mixed with 10 7 Balb/c x C57 alloactivated lymphocytes cells, or mixed with 10 6 IL-4 secreting J588L lymphoma cells (allogeneic to C57)
  • the alloactivated cells were prepared by culturing Balb/c splenocytes with C57 splenocytes at a ratio of 10 1 at 3 x 10 6 /mL in RPMI 10% FCS for 3 days All cells were washed in PBS and injected subcutaneously in the flanks of naive C57 mice Mice were observed for tumor formation, and sacrificed after tumors reached 1 cm in diameter Mice treated with IL-4 secreting cells survived significantly longer than the other groups with 2 out of 3 long term survivors The group treated with alloactivated cells alone had no long term survivors
  • mice showing various degrees of activation are tested in the mouse lymphoma treatment model
  • Mixed lymphocyte cultures are set up using splenocytes from a variety of inbred mouse strains at a 10 1 responder stimulator cell ratio
  • cultures are set up using a particular responder stimulator strain combination, but at different cell ratios
  • the activity is measured in XTT Formazan assay and esterase assay
  • the cultured cells are supplemented with additional splenocytes, as necessary, to normalize the cell ratio, and admixed with 1 x 10 s live or irradiated J588L lymphoma cells
  • the preparation is the injected into Balb/c mice, and the effect on survival is monitored
  • the mice can be rechallenged with a subsequent dose of live lymphoma cells to test for a persisting immunological response.
  • the survival data is then correlated with the functional activity measured during the culture period.
  • Standard cultures containing C57:Balb/c splenocytes (10:1) are compared for efficacy in the mouse lymphoma model with cultures containing: a) C57:Aj:Balb/c splenocytes (9:1 :1 or 5:5:1); b) C57:Aj:C3H splenocytes (9:1:1 or 5:5:1); c) C57:Aj:C3H:Balb/c splenocytes (8:1 :1:1 or 3:3:3:1).
  • EXAMPLE 5 EXPERIMENTS WITH CULTURED HUMAN CELLS
  • the degree of alloactivation (a potential reflection of potency in therapy) can be measured according to the functional assays detailed in Example 3. This example illustrates the degree of activation revealed by the assays.
  • Human peripheral blood monocytes were isolated from samples taken from a number of unrelated human volunteers, and set up in one-way mixed lymphocyte cultures at a 10:1 responderstimulator ratio as described elsewhere in this disclosure. The assays were run after 2-3 days in culture. The results are shown in Figures 3 and 4. Each of the individuals is indicated by a unique letter, with the responder cells being indicated before the stimulator cells. Thus, the designation A x B means that cells from individual A were cultured with inactivated cells from individual B.
  • esterase activity can also be measured by flow cytometry using the esterase substrate, fluorescein diacetate (FDA). T cells with high esterase activity can be identified by Phycoerythrin-labeled CD3 antibody in conjunction with FDA. These measures correlate well with blastogenesis (determined after culturing for one week), or the level of IL-2 or IFN- ⁇ in the supernatant.
  • FDA fluorescein diacetate
  • Allo-activated human lymphocyte cultures were produced using cells from either one, two, three or four unrelated donors. 3 x 10 s cells/mL were cultured in 2% FCS-RPMI at 37°C for 2 days. Two-donor populations were produced by admixing responder cells with stimulator cells at a 10:1 ratio. Populations containing three or four donor cells were produced by mixing responder ceils with two or three different stimulator cells at ratios of 9:1 :1 or 8:1 :1 :1.
  • Figure 5 shows the characteristics of the cells measured using flow cytometry. All values represent percentage of brightly fluorescent cells after counting 4000 cells on a Coulter EPICS XL Cytometer.
  • Histamine is known to induce the activity of T suppressor cells. Since T suppressor cells can play a role in controlling the activity of the MLR, the effect of histamine and of a potent histamine type 2 (H2) receptor blocking drug, Cimetidine, was tested in alloreacting cell cultures. Cell populations composed of alloactivated human peripheral blood mononuclear cells were produced using cells from unrelated donors. All cultures contain a 10:1 ratio of responder:stimuiator mononuclear cells at 0.5 x 10 s cells/mL. In some cultures, 20 ⁇ g/mL histamine or 20 ⁇ g/mL
  • Cimetidine were added on day 0.
  • Figure 7 shows the results measured using a Formazan reduction (XTT) assay. Histamine induced suppression and decreased strength of the allo-activation. Cimetidine enhanced activity, possibly by blocking the development of suppression.
  • XTT Formazan reduction
  • This example describes animal experiments in which immunological treatment of established malignant tumors leads to tumor regression and induction of permanent, long lasting tumor specific immunity.
  • the tumor used in this study is a non-immunogenic glioma histocompatible with the Fischer 344 (F344) rat, designated RT-2 (also known as D74).
  • D74 is an extremely aggressive, transplantable tumor in the F344 rat with histologic and clinical characteristics of Glioblastoma
  • Multiforme It is essentially incurable by standard therapeutic protocols. Intracranial implantation of as few as 10 cells results in fatal brain tumors in about 40 days. When injected subcutaneously, as few as 500,000 ceils form progressively growing tumors, first palpable in about 5 days, then progressing to large, 2 to 3 cm tumors in 3 to 4 weeks. D74 is not immunogenic on its own, as inoculation of naive rats with multiple doses of large numbers of lethally irradiated (10,000 rads) D74 tumor cells does not confer immunity. Surgical removal of well established growing tumors also does not result in subsequent immunity of the host.
  • Allogeneic cells were sensitized by in vitro mixed lymphocyte culture (MLC) in the following manner.
  • Spleen cells for use as a source of responder or stimulator lymphocytes were aseptically removed and minced into single cell suspensions in phosphate-buffered saline (PBS). The cells were passed through fine mesh gauze to remove small particulate debris, and washed twice by centrifugation (1500 rpm). The stimulator cells were inactivated by irradiation with 3000 Rads using a Cs 137 source.
  • Responder cells were cultured at 3 million/mL in RPMI-1640 containing 10% fetal calf serum, antibiotics (streptomycin/penicillin) and 5 x 10 "5 M ⁇ -mercaptoethanol; then stimulated with irradiated spleen cells at a 5:1 responderstimulator cell ratio. After 3 days at 37°C, the cells were harvested by centrifugation, washed twice in PBS, and suspended in PBS at 500 million/mL. This preparation is referred to in this example as a cytoimplant.
  • Cytoimplants were administered to F344 rats bearing established (4 to 7 mm) D74 tumors growing in the left thigh. The tumors were initiated approximately 10 days earlier by injecting naive F344 rats subcutaneously with 0.5 million D74 cells suspended in 100 ⁇ L PBS. Cytoimplants were suspended in a tuberculin syringe fitted with a 25 gauge needle, and were injected directly into the tumor nodule in a volume of about 100 to 250 ⁇ L. Tumor sizes were measured bidirectionally using calipers 2 to 3 times/week, until the tumors reached 3.0 cm, at which time the animals were sacrificed.
  • tumor diameters were as follows Group 1 (saline control) 20 mm (representing growth of ⁇ 2 mm per day), Group 2 10 mm (down from 12 5 mm on Day 20, statistically significant with respect to Group 1) Group 3 14 mm (statistically significant with respect to Group 1), Group 4 15 mm (down from 17 mM on Day 21 , with at least one animal showing signs of regression and three showing signs of stabilization), Group 5 15 mm (with several animals still showing some evidence of tumor progression)
  • lymphocytes from a first subject that are alloactivated against leukocytes from a second subject can be used to treat established tumors of a third subject by implantation into the tumor bed
  • results in individual subjects treated by the same protocol are heterogeneous Sequential implants have a synergistic effect, and can lead to not only extended survival, but also tumor regression Implant cells work well when they are cultured with stimulator cells that are MHC incompatible, and certain donor combinations seem to work better than others
  • Tumor cells are obtained during surgical resection of the primary neoplasm, and cryopreserve at the time of surgery The tumor cells are proliferated ex vivo if necessary to obtain sufficient cells for the anticipated course of therapy Thawed or cultured tumor cells are subjected to 10,000 rads of gamma irradiation Preparative-scale mixed lymphocyte cultures using inactivated patient stimulator cells and donor leukocytes are conducted generally as described in Example 1
  • the mononuclear cells used to prepare each cellular vaccine are obtained from healthy, unrelated donors Donors are prescreened to minimize risk for infectious diseases as described in Example 7, and those that test positive are eliminated. By using genetically disparate donors, the likelihood of hyperacute rejection of the second administration is decreased.
  • the mixed lymphocyte culture is conducted by mixing donor and inactivated patient peripheral blood mononuclear cells at a ratio of 10:1, and culturing at 3 x 10 6 cells/mL in AIMV supplemented with 2% fetal calf serum for 3 days at 37°C.
  • the total number of mononuclear cells required for a single inoculum is no more than 1 x 10 9 .
  • the stimulated cells are collected and washed by centrifugation, then suspended in sterile, injectable saline. Quality control of the production of activated cells includes monitoring cell counts and viability, testing for mycoplasma and endotoxin, and monitoring for lymphocyte activation using early activation markers, as described in Example 7. Before use in treatment, the alloactivated cell preparation is also evaluated according to functional release criteria.
  • the Tetrazolium Reduction Assay (XTT) described in Example 3 is conducted on a cell sample. Flow Cytometry is conducted to measure cell surface expression of CD69 using fluorescent antibody; or increased intracelluiar esterase activity using fluorescein diacetate.
  • Cultured cells are considered to be sufficiently activated if the level measured in either one (but preferably both) of these assays is > 10% above unstimulated donor control value on any day of the culture period (day 1 , day 2, or day 3). Once the culture passes the criteria, testing on subsequent days is not needed. The cells are harvested on day 3, mixed with the requisite number of primary or cultured tumor cells, and prepared for human administration.
  • the vaccine composition consists essentially of an alloactivated cell population mixed with tumor cells. Patients receive one of three different doses: 1 x 10 8 MLC cells; 3 x 10 8 MLC ceils; or 1 x 10 9 MLC, mixed with up to 1 x 10 7 inactivated tumor cells, depending on availability. The same dose is given four times on a weekly schedule.
  • MTD maximum tolerated dose
  • a comparison is made between the 4-week vaccination schedule alone, and a vaccination course initiated by direct implantation into a tumor mass.
  • the implant group is treated two days to a week after colectomy, using ultrasound to guide an injection needle into a sizeable metastatic tumor mass in the liver.
  • the metastatic site is injected with a preparation of 10 x 10 9 MLC alloactivated cells alone, suspended in a minimum volume of saline. Beginning one week later, the patients in this group also receive the 4-week course of the MLC- tumor cell vaccine.
  • Safety of the compositions is monitored by several criteria, including local induration, pruritus, or necrosis at the injection site; systemic effects such as fever, malaise, headache, and altered hematological or renal parameters.
  • the presence of a cellular immune response in the treated patient can be monitored by several criteria.
  • Patient lymphocytes obtained before and after each inoculation are cultured with irradiated allogeneic cells of donor origin or from a third party (for anti-allotype response), or irradiated patient tumor cells, or third-party tumor cells (for specific anti-tumor response).
  • the response of patient lymphocytes in culture is determined by measuring proliferation using reduction of MTT or one of the other functional assays as a surrogate marker for cellular division.
  • Expression of CD69 is determined by immunofluorocytometry using PE-labeled antibody.
  • the responding T cells are costained for CD4, CD8, or CD31 to identify helper or suppressor subsets, or for CD45RF to distinguish T H1 from T H2 cells.
  • Cytokines IL-2, IL-4, IFN- ⁇ and TNF- ⁇ secreted into the culture media are quantified by ELISA.
  • IL-2 and IFN- ⁇ correlate with T H1 activity
  • IL-4 correlates with T H2 activity
  • TNF- ⁇ correlates with the activity of both.
  • Patients' PBL are also optionally tested for their ability to respond to autologous tumor cells in culture.
  • General T cell activation can be measured by the functional assays described in Example 3, [ 3 H] thymidine incorporation, or blastogenesis.
  • Cytotoxic T cell activity can be measured as cytolysis of 51 Cr labeled tumor ceils.
  • the effective delayed type hypersensitivity (DTH) anti-tumor response in the treated patient is measured by comparing the 48-hour response of the intradermal administration of 5 x 10 5 autologous tumor cells, mumps, tricophyton, or PPD antigens with that observed for the same series before treatment.
  • DTH delayed type hypersensitivity
  • the patients are monitored for the extent of the clinical and immunological response for at least three months following therapy.
  • Clinical criteria is monitored, in part, by tracking the volume of tumor metastasis present in the liver.
  • a CT scan is performed at regular intervals, the volume of each metastatic site is calculated, and the volumes are compared with the measurements obtained before treatment.
  • Progression of disease is indicated by an increase in volume of the metastasis, or an increase in the number of metastatic sites.
  • a successful outcome is indicated by reversal of the disease, or slower progression in comparison with the typical outcome for patents with colon cancer of the same grade.
  • This protocol describes the overall approach to production of the mixed lymphocyte culture.
  • the design of this methodology takes into account Good Manufacturing (GMP) and Good Laboratory (GLP) Practices, and complies with requirements of Code 21 of U.S. Federal Regulations.
  • Patient peripheral blood mononuclear cells at least 2 x 10 9 cells are collected by modified leukapheresis from the patient to be treated. Isolation of cells is performed on a Baxter Fenwall apheresis machine or equivalent machine using the Stem Cell Collection Procedure. Cells are shipped in a Baxter-type component bag on ice (4-10°C). Transit temperature is monitored using MONITOR-MARKTM Time/Temperature Tags.
  • Donor peripheral blood mononuclear cells are collected by modified leukapheresis from a healthy individual. Isolation of cells is performed on a Baxter Fenwall apheresis machine or equivalent , using the Stem Cell Collection Procedure. Donors are unrelated, anonymous, and random individuals, picked from a list of prescreened potential donors.
  • Prescreening of the donors should indicate negative risk factors for HIV, Hepatitis, Spongioform Encephalitides, or Tuberculosis.
  • Each cell component is tested negative for HIV 1/2 Ab, HIV Ag, CMV Ab, HTLV l/ll Ab, HCV Ab, HBcAb, HBsAg and RPR.
  • Cells are shipped in a Baxter-type component bag on ice (4-10°C).
  • each component Upon receipt each component is tested for sterility, appropriate cell counts, and viability.
  • Components are maintained at 4-10°C until use, and used or frozen within 72 hours of collection.
  • Thawed frozen material are used within 2 hours and not re-frozen. Pre-clinical studies indicate that components stored at 4° C in ACD anticoagulated plasma or material frozen in DMSO-containing media are suitable for the production of effective cell compositions.
  • Plasma is removed form both the donor and patient components by centrifugation.
  • Donor plasma may be collected and heat-inactivated for use as a medium supplement.
  • Component cells are suspended in small volumes of PBS and appropriate volumes of each suspension is mixed to produce a culture that contains 3 x 10 6 mononuclear cells/ml in AIMV medium at a ratio of 10:1 to 20:1 (donor: patient cells).
  • Heat-inactivated donor plasma is added to a final concentration of 2%.
  • Mixed cells are pumped into Fenwall 3 liter gas permeable culture bags through the use of the Fenwall solution pump and sterile set-up. Samples of the component cells may also be set up in small culture tubes for testing of lymphocyte activation. Testing of functional activity is compared with control cultures containing unstimulated donor cells alone. Cell mixtures are cultured in a ISO-°9000 Forma 37°C incubator with 5% humidified and
  • the ceil preparation is suspended in sterile 25% human albumin, and placed in sterile injectable vials for transport. Each preparation is labeled with an expiration date and time, which is 30 hours after packaging, and accompanied by appropriate instructions, release specification results, and a MONITOR-MARKTM Time/Temperature Tag. Cell preparations are packaged and shipped via overnight courier service. If not used immediately, the cells are stored in a refrigerator at 4-10°C. Any preparation not implanted before the expiration date is discarded. In process tests that measure product consistency include:
  • Cells must also meet satisfactory functional criteria. Preparations not meeting any of these criteria are not used for treating patients.
  • Patient may be positive for HBcAb or CMV Ab, and components are labeled as such. If CMV negative donor components are not available, a CMV Ab positive donor component may be used, even for CMV negative patients.

Abstract

L'invention concerne des procédés et des compositions qui permettent de traiter une tumeur par implantation d'une population cellulaire alloactivée à proximité de la tumeur. La population cellulaire est constituée d'une pluralité de cellules donneuses tierces cultivées ensemble ex vivo et récoltées presque au moment de la sécrétion maximale de cytokine. Une fois placées dans le lit de la tumeur, les cellules alloactivées sollicitent une participation active de l'hôte qui réagit alors contre la tumeur et fournit un niveau de protection continue. L'utilisation de multiples cellules donneuses tierces confère des avantages particuliers en termes d'efficacité, de délai et de facilité d'emploi.
PCT/US1998/021413 1997-10-10 1998-10-09 Implants comprenant des combinaisons de cellules allogeniques pouvant etre utilisees dans le traitement du cancer WO1999018981A1 (fr)

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DE69837552T DE69837552D1 (de) 1997-10-10 1998-10-09 Allogene zellkombination enthaltende implantate zur krebsbehandlung
EP98952216A EP1021197B1 (fr) 1997-10-10 1998-10-09 Implants comprenant des combinaisons de cellules allogeniques pouvant etre utilisees dans le traitement du cancer
AU97969/98A AU9796998A (en) 1997-10-10 1998-10-09 Implants comprising combinations of allogeneic cells for use in cancer treatment
CA002346769A CA2346769C (fr) 1997-10-10 1998-10-09 Implants comprenant des combinaisons de cellules allogeniques pouvant etre utilisees dans le traitement du cancer

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US11173205B2 (en) 2014-11-05 2021-11-16 Memorial Sloan Kettering Cancer Center Methods of selecting T cell line and donor thereof for adoptive cellular therapy

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US7361332B2 (en) * 1995-03-17 2008-04-22 The Regents Of The University Of California Treating tumors using implants comprising combinations of allogeneic cells
US6719805B1 (en) * 1999-06-09 2004-04-13 C. R. Bard, Inc. Devices and methods for treating tissue
US7332158B2 (en) * 2002-05-29 2008-02-19 Demao Yang Compositions and treatments for myelosuppression by ex vivo activated immune cells
US7048922B2 (en) * 2002-05-29 2006-05-23 Demao Yang Stimulation of hematopoiesis by ex vivo activated immune cells
US20060057121A1 (en) * 2004-09-10 2006-03-16 Demao Yang Compositions and treatments using ex vivo activated cells for myelosuppressed patients
US20080089875A1 (en) * 2006-10-13 2008-04-17 Zheng Cui Methods and compositions for the treatment of cancer
US8455188B2 (en) * 2007-01-26 2013-06-04 University Of Louisville Research Foundation, Inc. Modification of exosomal components for use as a vaccine
TW201034641A (en) * 2009-02-28 2010-10-01 Charles Knezevich Apparatus, system, and method for creating immunologically enhanced spaces in-vivo
JP2013544600A (ja) * 2010-11-23 2013-12-19 プレサージュ バイオサイエンシズ，インコーポレイテッド 固体デリバリーのための治療方法および組成物
US10035009B2 (en) 2013-04-15 2018-07-31 The Board Of Trustees Of The Leland Stanford Junior University Systems and methods for treating pancreatic cancer
US20210196337A1 (en) * 2019-12-26 2021-07-01 Jerome Canady Research Institute for Advanced Biological and Technological Sciences Method for treatment for combination cold atmospheric plasma therapy of solid tumors

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